Allergies are caused by a dysfunction in the immune system, which reacts to harmless proteins, mainly contained in pollens, mites, epithelia and foodstuffs, by producing IgE-class antibodies.
Recent estimates indicate that over 25% of the population of the industrialised countries suffers from this diseases which, if it persists, can lead to a deterioration in the symptoms (e.g. appearance of asthma) and sensitisation to other allergens, thus complicating the choice of the most suitable treatment.
About a third of all the allergic subjects in the world are allergic to tree pollen. In temperate regions, pollen from trees belonging to the order Fagales (birch, alder, hazel, oak and hornbeam) is one of the major causes of allergic asthma and rhinitis. About 95% of patients allergic to birch pollen produce IgE antibodies against Bet v 1, and 60% of these patients only react to Bet v 1 (cDNA deposited at GenBank acc. no. X15877), the major allergen of birch pollen (1).
A high percentage of individuals allergic to birch pollen (50%-93%) develop allergic reactions after eating certain foods (apples, carrots, hazelnuts and celery). This type of hypersensitivity in patients with birch pollen allergy, characterised by allergy to fruit, nuts and vegetables, is described as “pollen-food syndrome” (PFS). The symptoms range from reactions of the mucosa of the upper digestive tract (oral allergy syndrome) and the gastroenteric tract to urticaria and asthma, and in some cases can cause anaphylactic shock. Adverse reactions to fruit occur most frequently with apple. In the case of pollinosis caused by birch pollen, this syndrome is mainly mediated by cross-reacting IgE antibodies originally induced by Bet v 1 (2).
The treatment of these patients is mainly based on food allergen avoidance from the diet. For the treatment of allergy to pollens, animals and mites, hyposensitizing specific immunotherapy (SIT), unlike pharmacological treatment, has proved to be an effective form of etiological treatment, which positively affects some immunological parameters that are the basis of the disease. SIT involves administering increasing doses of standardised extracts (vaccines) obtained from the substance that causes the disease (3). In this way a kind of “immunological tolerance” against that substance is gradually induced in the patient, accompanied by a reduction in, if not the disappearance of, the allergy symptoms. Conversely, specific immunotherapy is not commonly used to treat food allergies in view of the high risk of inducing serious side effects, including anaphylactic shock.
Although the cross-reactivity between Bet v 1 and homologous food proteins underlies the reactions induced by PFS, it has been found that SIT with birch pollen does not induce any improvement in food allergy symptoms.
This inefficacy has been observed in the case of carrot allergy, mainly caused by the allergen Dau c 1, a homologue of Bet v 1, in which primary sensitisation arises towards the birch allergen and subsequent sensitisation, after eating carrot, towards new epitopes of Dau c 1 which do not cross-react with Bet v 1 (4).
In the treatment of apple allergy, SIT with birch pollen extract gave rise to controversial, sometimes favourable results, probably due to the greater sequence identity between Bet v 1 and Mal d 1 (60% of the amino-acid sequence, as against 40% for Dau c 1).
In a 1998 study, in 84% of patients allergic to birch and apple who were treated with SIT based on birch pollen extract, a significant reduction (50-95%) or total disappearance of OAS (oral allergy syndrome) symptoms after eating apple had already disappeared after the first year's treatment (5). In a more recent study, published in 2004, SIT with birch pollen extract led to a significant reduction in SPT reactivity to Bet v 1 and Mal d 1 after only three months' treatment. The IgG4 antibodies strongly induced against Bet v 1 exhibited cross-reactivity to Mal d 1, thus supporting the hypothesis that SIT against birch pollen allergy can also reduce allergy to foods containing Bet v 1 homologous allergens (6). Another study published in the same year (2004) showed, in patients allergic to birch and apple, that SIT based on birch pollen extract significantly improved the allergic symptoms caused by pollen, but did not reduce the severity of apple allergy (7).
On the basis of current knowledge, it therefore cannot be stated with certainty that immunotherapy with birch pollen allergens provides significant benefits in the case of allergy to foods containing Bet v 1 homologues, which should therefore be treated as a separate disease.
One of the allergens most frequently involved in PFS cross-reactivity to Bet v 1 is Mal d 1, the major allergen of apple, a fruit belonging to the Rosaceae family. Mal d 1 shares approximately 60% of its amino-acid sequence with Bet v 1 (identity), and cross-reactivity experiments have demonstrated the presence of common IgE and T epitopes (8,9).
Mal d 1 (Acc. No. Q9SYW3 or No AJ417551) is a 159 amino-acid protein with a molecular weight of 17.7 kDa. (10). This allergen belongs to the PR-10 family of “pathogenesis related proteins”, i.e. ubiquitous proteins produced by plants in response to environmental or pathological stresses, the function of which is believed to be connected with steroid transport. Mal d 1 is located in both the peel and the pulp of apples. Measurement of Mal d 1 content in protein extracts from numerous varieties of apple demonstrate that this allergen is present in considerably different concentrations in the different varieties; variability in the content of Mal d 1 was also found in the same variety grown in different places. Moreover, even in varieties with a low Mal d 1 content, it has been observed that the allergen concentration increases significantly during ripening and storage of the fruit.
Mal d 1 is represented by a gene family with at least eighteen members, characterised by the presence or absence of introns in the gene. Some members are highly conserved in the different apple varieties (Mal d 1.01, Mal d 1.02), while others present more variability (Mal d 1.04, Mal d 1.05, Mal d 1.06 A, B, C) (11). When SPT analysis was conducted on patients with pollen-food syndrome, an association between the protein variants encoded by genes Mal d 1.04 and Mal d 1.06 A and greater allergenicity of the apple varieties was observed. The results of this type of study may have applications in the identification and cultivation of apple varieties with lower allergenicity for use as a food or as raw material for conventional immunotherapy.
The development of hypoallergenic food allergens for use in specific immunotherapy may represent a good alternative to food allergen avoidance and allow the treatment of a disease which, as well as involving high risks for patients' health, has a significant negative impact on quality of life and can cause incapacitating nutritional imbalances.
In recent years, a great deal of attention has focused on developing safer, more effective vaccines, consisting of recombinant proteins mutagenised at the level of amino acids important for IgE binding, namely hypoallergenic variants that favourably influence the natural progression of the disease without causing adverse effects (12).
Some studies of hypoallergenic variants of Mal d 1 or homologous proteins from other fruit belonging to the Rosaceae family are available in the literature.
On the basis of the cross-reactivity between Bet v 1 and Mal d 1, a mutant of Mal d 1, the major apple allergen, was produced by site-directed mutagenesis of five amino acid residues on isoform Mal d 1.0108 (T10P; 130V, T57N, T112C and I113V) selected by analogy with a hypoallergenic mutant of Bet v 1 allergen (9). The substitution of said amino acids reduced the allergenic activity of Mal d 1 by 90%, as demonstrated by SPT and DBPCFC (double-blind placebo-controlled food challenge) analysis. The same mutant with five amino-acid substitutions was tested in parallel with the analogous mutant of Bet v 1 and with the corresponding wild-type molecules (13). Although the sera of patients allergic to birch and apple exhibited greater IgE reactivity against Bet v 1 in immunoblotting tests, a reduction in binding to both the mutants tested was observed. In ELISA assays, in the majority of sera tested (10/14), a reduction from 30 to 88% in the IgE-binding of mutant Mal d 1 was observed compared to the wild-type allergen. However, the mutagenesis of the five amino acids selected does not seem to prejudice the IgE reactivity of the main epitopes of Mal d 1, as no variation in specific IgE binding was observed in some of the sera analysed (3/14).
In Pru av 1, the major cherry allergen homologous to Mal d 1, punctiform substitution of serine 112 proved critical for recognition of the molecule by IgEs. The mutagenesis of the same amino acid in proline in homologue Bet v 1 confirmed the importance of serine 112 in preserving the structure of the cross-reactive IgE epitope. The substitution of Glu45 by tryptophan in the P-loop sequence of Pru av 1 demonstrated that this region is an IgE epitope cross-reactive to Bet v 1 (14). Three other Pru av 1 variants were obtained by mutagenesis of amino acids in position 28 (Asn28Lys), 108 (Pro108Ala), or both. Reduced IgE binding in up to 80% of sera from patients allergic to birch and cherry was observed for the single mutant (Asn28Lys) and the double mutant (Asn28Lys, Pro108Ala), whereas a reduction in only 12% was obtained for the single mutant on Pro108, suggesting that amino acid Asn28 in Pru av 1 is involved in an IgE epitope. This asparagine is exposed to the solvent, and is part of one of the areas proposed as the main antigen sites in homologous protein Bet v 1 (15).
The study of the three-dimensional structure of Bet v 1 by NMR analysis and X-ray diffraction led to the identification of three zones with an area exceeding 600 Å which may be involved in IgE binding (16). These three zones exposed on the surface consist of highly conserved amino acids in the homologous allergens expressed in species belonging to the order Fagales, and have been proposed as potential IgE epitopes responsible for the cross-reactivity between Bet v 1 and the homologous proteins of plant pollens. Site-directed mutagenesis of amino acids in these areas confirmed their involvement in IgE binding. The mutations in Bet v 1 that characterise the two multiple mutants (T28, Q32, S45, G108) and (V5, S42, S45, K78, V103, I123, E134, H156, N160), for example, modify up to five different areas distributed on the molecular surface, including the three zones described above, and cause a reduction in allergenicity (17). The amino-acid sequences that constitute the three IgE epitopes proposed by Gajhede are also conserved in the food allergens homologous to Bet v 1. In order to reduce surface similarity to Bet v 1, the conserved amino acids Thr 28, Gln 32 and Ser 45 of Mal d 1, located in one of the areas described above, were substituted with amino acids not present in Bet v 1 in the corresponding positions. The substitution of these residues did not vary the ability of mutant Mal d 1 to inhibit IgE-Bet v 1 binding compared with the wt counterpart, while histamine release was abolished in basophils from the blood of the same birch allergic patients in only one case out of the five tested (24).
In order to identify the IgE epitopes involved in the clinical symptoms of pollen-food syndrome, a chimeric protein was generated by grafting four short peptide stretches of Mal d 1 onto Bet v 1 sequence (18). The transplanted regions included the amino acid residues previously proved crucial for patients' IgE binding to both allergens, Bet v 1 and Mal d 1: T10, F30, S57, S112, I113 and D125. The IgE reactivity of the chimeric protein was tested using sera from two groups of patients with birch allergy, without PFS or showing PFS symptoms following apple ingestion. The chimeric molecule was recognised by the IgEs of both groups, but binding was significantly lower in the group with no PFS compared to patients with apple allergy, suggesting that the grafted sequences on Bet v 1 were involved in Bet v 1/Mal d 1 IgE cross-reactivity.
The birch pollen allergy characterised by PFS represents an excellent study model for identifying cross-reacting IgE epitopes and allowing the production of recombinant allergens with a reduced IgE binding capacity.
The possibility to use engineered multimeric molecules consisting of different allergens from the same organism or different organisms for specific hyposensitizing therapy has long been considered fascinating. This approach allows a number of allergens to be assembled in a single molecule with the advantage of producing a single preparation containing the allergens in a precise molar ratio. The association of Bet v 1 and Mal d 1 in a single hybrid molecule should represent the T-epitope repertoire of both molecules, and may induce a strong IgG protective response (17) against both allergens. Induction of IgG antibodies specific for the sensitising allergen is one of the factors correlated with the benefit induced by SIT. Such (protecting) antibodies can inhibit the IgE binding to the antigen, altering the tridimensional conformation of the molecule.
Some studies have been conducted to investigate the clinical effects on birch pollen allergy of SIT with trimeric derivatives of the allergen Bet v 1 (19). The treatment induced strong IgG1 and IgG4 specific allergen antibody response and reduced nasal and skin reactivity, but did not yield to a significant improvement in clinical symptoms.
Significant IgG antibody response was also observed in experiments in mice treated with the dimeric wt or mutant form in position 112 of carrot allergen Dau c 1, homologous to Bet v 1 allergen (20). Both dimeric variants proved more antigenic than the mixture of the corresponding monomeric forms. Moreover, all the murine sera produced against Dau c 1 (in monomeric, dimeric, wt and mutant form) contained specific antibodies cross-reacting to epitopes recognised by human IgE, indicating that structurally altered antigens like dimers are able to induce the production of antibodies specific for conformational epitopes.
The use of a hypoallergenic hybrid variant like Bet v 1-Mal d 1 may eliminate the difficulties caused by severe adverse reactions to food allergens and improve a tolerogenic response not only to Bet v 1, as occurs when SIT with birch pollen extract is used, but also to the major allergen of apple.